1. Field of the Invention
The present invention relates to a microorganism biocontrol agent, in particular to a fungi transformant containing melanin biosynthesis genes to increase the tolerance to environmental stress and its method.
2. The Prior Arts
Beneficial microorganisms such as Metarhizium spp., Beauveria spp., Trichoderma spp. and Bacillus spp. have been applied in pest management for a long time. There are numerous studies showing positive effects in pest control with beneficial microorganisms in field application so far (Genthner et al., 1998). However, the stability and reproducibility of the studies were limited in field application due to the factors of stressful environmental condition. (Alves et al., 1998; Hedgecock et al., 1995). Natural environmental factors such as high temperature, drought and UV radiation are the bottle necks for field application (Fargues, 1996). Therefore, it becomes an important issue to protect the beneficial microorganisms to lower the stress of the environment.
The survival of biocontrol microorganisms in field during the contact and application period for the strain of biological control microorganism and the targets (such as pest or pathogens) is important for tolerance of stressful environment. The application effects won't be stable for those that can not overcome the barriers and survive in field. Therefore, the improvement of tolerance to stressful environment for the biocontrol microorganism is an important issue. At present, the improvement is employed through formulation. Hedimbi et al (2008) used olive oil containing commercial sunscreens as additive to treat Metarhizium anisopliae conidia, and exposed them to an artificial UV source for up to 5 hours. Survival of conidia in oil formulation was around 29-40% while in control (water) was 4%. The conidial germination rates produced from mycelium were lowered to 20% when irradiated with UV-A for 6 hours (Rangel et al. 2008). The increased osmotic stress also made the germination rate of M. anisopliae dropped sharply. Bacillus thuringiensis is a biocontrol agent other than eukaryotes. The δ-endotoxin produced in B. thuringiensis is easily degraded by UV-radiation. The activity of B. thuringiensis is lost under short term exposure of sunshine. Previous studies have shown that the UV tolerance was increased in melanin producing B. thuringiensis strains. To overcome the stressful conditions mentioned above, the adjustment on application time (to avoid the strong light), formulation change (such as using oil to overcome the drought problem) or adding sunscreen to protect the microorganisms were employed. In addition, nutritional stress has been applied to increase the tolerance to high temperature, low water content and UV radiation. Trehalose and mannitol levels were accumulated in M. anisopliae conidia after nutritional stress, which may be the reason for high tolerance to the stressful condition (Rangel et al., 2008).
Biosynthesis of DHN melanin is synthesized by a polyketide pathway, through the genes encoding Polyketide synthase (PKS), Scytalone dehydratase (SCD), and 1,3,8-trihydroxynaphthalene reductase (THN). It started with a PKS using malonyl-CoA as a substrate to produce 1,3,6,8-tetrahydroxynaphthalene, 1,3,6,8-THN (Fujii et al., 2000), followed by reducatse catalysis to produce scytalone, dehydration by SCD to yield 1,3,8-trihydroxynaphthalene (1,3,8-THN), reduction by THN to yield vermelone, dehydration to produce melanin precursor 1,8-dihydroxynaphthalene (1,8-DHN), and then oxidation and polymerization to yield melanin.
Melanin is polymer existed broadly in organisms in nature and has a variety of biological functions (Hill et al., 1992). It is negatively charged, hydrophobic pigment with high molecular weight, which is formed by the oxidative polymerization of phenolic and/or indolic compounds. In many organisms, melanin protects cells from stressful conditions, such as oxidation, extreme temperature, UV radiation, chemical, and biochemical stresses (reviewed in Nosanchuk and Casadevall, 2003; Bell and Wheeler, 1986). Therefore, the use of melanin in the invention is a solution to protect biocontrol agents.